Understanding the mechanisms by which mucosal pathogens such as Vibrio cholerae colonize the human intestinal mucosa is key to the rational development of live-attenuated vaccine derivatives capable of inducing protective immunity against cholera and other enteric diarrheal diseases. Current parenteral vaccination strategies for these infections are largely ineffective. Although many V. cholerae genes required for intestinal colonization have been identified, the molecular mechanisms by which the proteins they encode promote vibrio adherence to host tissue is poorly understood. The studies described in this research proposal represent an attempt to understand at the molecular level, the contribution of the V. cholerae accessory colonization factor AcfB and AcfC proteins to the intestinal colonization properties of this emerging human pathogen. AcfB is a 75 kDa inner membrane protein that belongs to a large family of signal transducing proteins involved in bacterial chemotaxis. V.cholerae acfB mutants display an altered motility phenotype using a swarm plate motility/chemotaxis assay and produce reduced levels of cholera toxin and toxin-coregulated pilus. AcfC is a 26 kDa periplasmic protein that closely resembles bacterial sulfate binding proteins involved in solute transport and bacterial chemotaxis. Mutations within acfC specifically interfere with the ability of V. cholerae to migrate toward a gradient of galactose-6-sulfate in a standard chemotaxis assay. This proposal outlines a series of experiments aimed at understanding in molecular detail the contributions of the AcfB and AcfC proteins to vibrio chemotaxis/intestinal colonization. The long-term goal of these studies is to understand the structure and function of AcfB and AcfC so that we can use the information regarding the properties of these two proteins in the development of improved methods for treating and preventing cholera/enteric infections. There I are four specific aims in the present proposal: (1) chemotaxis/intestinal colonization/pilus production assays will be used to determine the features of AcfB that promote chemotaxis and/or pilus synthesis; (2) in vitro/in vivo model systems will define the features of AcfC required for binding galactose-6-sulfate and the contribution of this process to vibrio chemotaxis/intestinal colonization; (3) the infant mouse model of cholera infection and excised intestinal tissue will be used to determine the nature of the colonization defect in V. cholerae carrying mutations within acfBC genes; (4) recombinase-based in vitro expression technology (RIVET) will elucidate the role of AcfB in promoting maximal levels of pilus synthesis.